The present invention relates to novel molecules and, more particularly, to methods of treating MCP-1/CCR2 associated diseases, such as inflammatory diseases and cancer.
Over the past several years a growing number of leukocyte chemoattractant/activating factors (chemokines) were described [Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991); Schall and Bacon, Curr. Opin. Immunol., 6:865-873 (1994); Baggiolini, M., et al., Adv. Immunol., 55:97-1-79 (1994)]. Chemokines are produced and secreted by a wide variety of cell types in response to early inflammatory mediators such as IL-1β or TNF-α.
The chemokine superfamily comprises two main branches: the α-chemokines (also known as the CXC chemokines) and the β-chemokines (also known as the CC chemokines). This classification is based on whether the first two cysteines in the amino acid sequence are separated by a single amino acid (CXC) or are adjacent (CC). The α-chemokine branch includes proteins such as IL-8, neutrophil activating peptide-2 (NAP-2), melanoma growth stimulatory activity (MGSA/gro or GRO α), and ENA-78, each of which have both attracting and activating effects predominantly on neutrophils and T lymphocytes. The members of the β-chemokine branch affect other blood cell types such as monocytes, lymphocytes, basophils, and eosinophils [Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991); Baggiolini, M., et al., Adv. Immunol., 55:97-179 (1994); Miller and Krangel, Crit. Rev. Immunol., 12:17-46 (1992); Jose, P. J., et al., J. Exp. Med., 179:881-118 (1994); Ponath, P. D., et al., J. Clin. Invest., 97:604-612 (1996)], and include proteins such as monocyte chemotactic proteins 1-4 (MCP-1, MCP-2, MCP-3, and MCP-4), RANTES, and macrophage inflammatory proteins (MIP-1α and MIP-1β).
A third smaller branch of the chemokine superfamily comprises membrane-bound chemokines. Members of this class are designated CX3C chemokines [Bazan, J. F., et al., Nature 385:640-644 (1997)].
Chemokines can mediate a range of pro-inflammatory effects on leukocytes, such as triggering of chemotaxis, degranulation, synthesis of lipid mediators, and integrin activation [Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991); Baggiolini, M., et al., Adv. Immunol., 55:97-179 (1994); Miller, M. D. and Krangel, M. S., Crit. Rev. Immunol., 12:17-46 (1992)].
The chemokines bind to specific cell-surface receptors belonging to the family of G-protein-coupled seven-transmembrane-domain proteins [Murphy, P. M., Annu. Rev. Immunol., 12:593-633 (1994)] which are termed “chemokine receptors”. On binding their cognate ligands, chemokine receptors transduce an intracellular signal though the associated trimeric G proteins, resulting in, among other responses, a rapid increase in intracellular calcium concentration, changes in cell shape, increased expression of cellular adhesion molecules, degranulation, and promotion of cell migration.
Chemokines have been implicated as important mediators of allergic, inflammatory and autoimmune disorders and diseases, such as asthma, atherosclerosis, glomerulonephritis, pancreatitis, restenosis, rheumatoid arthritis, diabetic nephropathy, pulmonary fibrosis, and transplant rejection.
In particular, monocyte chemoattractant-1 (MCP-1), acting on its receptor CC Chemokine Receptor 2 (CCR-2), plays a role in a wide variety of indications. Upon binding to its receptor, MCP-1 induces a rapid increase in intracellular calcium concentration which leads to an increased expression of cellular adhesion molecules and eventually to cellular degranulation and the promotion of leukocyte migration.
Demonstration of the importance of the MCP-1/CCR-2 interaction has been provided by experiments with genetically modified mice. MCP-1−/− mice had normal numbers of leukocytes and macrophages, but were unable to recruit monocytes into sites of inflammation after several different types of immune challenge [Bao Lu, et al., J. Exp. Med. 1998, 187, 601]. Likewise, CCR-2−/− mice were unable to recruit monocytes or produce interferon γ when challenged with various exogenous agents; moreover, the leukocytes of CCR-2 null mice did not migrate in response to MCP-1 [Landin Boring, et al., J. Clin. Invest. 1997, 100, 2552], thereby demonstrating the specificity of the MCP-1/CCR-2 interaction. Two other groups have independently reported equivalent results with different strains of CCR-2−/− mice [William A. Kuziel, et al., Proc. Natl. Acad. Sci. USA 1997, 94, 12053, and Takao Kurihara, et al., J. Exp. Med. 1997, 186, 1757]. The viability and generally normal health of the MCP-1−/− and CCR-2−/− animals is noteworthy, in that disruption of the MCP-1/CCR-2 interaction does not induce physiological crisis. Taken together, these data suggest that molecules that block the actions of MCP-1 would be useful in treating a range of inflammatory and autoimmune disorders.
It is known that MCP-1 is upregulated in patients with rheumatoid arthritis [Alisa Koch, et al., J. Clin. Invest. 1992, 90, 772-779]. Moreover, several studies have demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating rheumatoid arthritis. A DNA vaccine encoding MCP-1 was shown recently to ameliorate chronic polyadjuvant-induced arthritis in rats [Sawsan Youssef, et al., J. Clin. Invest. 2000, 106, 361]. Likewise, inflammatory disease symptoms could be controlled via direct administration of antibodies for MCP-1 to rats with collagen-induced arthritis [Hiroomi Ogata, et al., J. Pathol. 1997, 182, 106], or streptococcal cell wall-induced arthritis [Ralph C. Schimmer, et al., J. Immunol. 1998, 160, 1466]. Perhaps most significantly, a peptide antagonist of MCP-1, MCP-1 (9-76), was shown both to prevent disease onset and to reduce disease symptoms (depending on the time of administration) in the MRL-1 pr mouse model of arthritis [Jiang-Hong Gong, et al., J. Exp. Med. 1997, 186, 131].
MCP-1 is also upregulated in atherosclerotic lesions, and it has been shown that circulating levels of MCP-1 play a role in disease progression [Abdolreza Rezaie-Majd, et al, Arterioscler. Thromb. Vasc. Biol. 2002, 22, 1194-1199]. MCP-1 is responsible for the recruitment of monocytes into atherosclerotic areas, as shown by immunohistochemistry of macrophage-rich arterial wall [Yla-Herttuala et al., Proc Natl Acad Sci USA 88:5252-5256 (1991); Nelken et al., J Clin Invest 88:1121-1127 (1991)] and anti-MCP-1 antibody detection [Takeya et al., Human Pathol 24:534-539 (1993)]. LDL-receptor/MCP-1-deficient and apoB-transgenic/MCP-1-deficient mice show significantly less lipid deposition and macrophage accumulation throughout their aortas compared with wild-type MCP-1 strains [Alcami et al., J Immunol 160:624-633 (1998); Gosling et al., J Clin Invest 103:773-778 (1999); Gu et al., Mol. Cell. 2:275-281 (1998); Boring et al., Nature 394:894-897 (1998)].
It is known that MCP-1 is upregulated in human multiple sclerosis, and it has been shown that effective therapy with interferonβ-1β reduces MCP-1 expression in peripheral blood mononuclear cells, suggesting that MCP-1 plays a role in disease progression [Carla Iarlori, et al., J. Neuroimmunol. 2002, 123, 170-179]. Other studies have demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR-2 interaction in treating multiple sclerosis; all of these studies have been demonstrated in experimental autoimmune encephalomyelitis (EAE), the conventional animal model for multiple sclerosis. Administration of antibodies for MCP-1 to animals with EAE significantly diminished disease relapse [K. J. Kennedy, et al., J. Neuroimmunol. 1998, 92, 98]. Furthermore, two more recent reports have now shown that CCR-2−/− mice are resistant to EAE [Brian T. Fife, et al., J. Exp. Med. 2000, 192, 899; Leonid Izikson, et al., J. Exp. Med. 2000, 192, 1075].
It is known that MCP-1 is upregulated in patients who develop bronchiolitis obliterans syndrome after lung transplantation [Martine Reynaud-Gaubert, et al., J. of Heart and Lung Transplant., 2002, 21, 721-730; John Belperio, et al., J. Clin. Invest. 2001, 108, 547-556]. In a murine model of bronchiolitis obliterans syndrome, administration of an antibody to MCP-1 led to attenuation of airway obliteration; likewise, CCR2−/− mice were resistant to airway obliteration in this same model [John Belperio, et al., J. Clin. Invest. 2001, 108, 547-556]. These data suggest that antagonism of MCP-1/CCR2 may be beneficial in treating rejection of organs following transplantation.
Other studies have demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating asthma. Sequestration of MCP-1 with a neutralizing antibody in ovalbumin-challenged mice resulted in marked decrease in bronchial hyperresponsiveness and inflammation [Jose-Angel Gonzalo, et al., J. Exp. Med. 1998, 188, 157]. It proved possible to reduce allergic airway inflammation in Schistosoma mansoni egg-challenged mice through the administration of antibodies for MCP-1 [Nicholas W. Lukacs, et al., J. Immunol. 1997, 158, 4398]. Consistent with this, MCP-1−/− mice displayed a reduced response to challenge with Schistosoma mansoni egg [Bao Lu, et al., J. Exp. Med. 1998, 187, 601].
Other studies have demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating kidney disease. Administration of antibodies for MCP-1 in a murine model of glomerularnephritis resulted in a marked decrease in glomerular crescent formation and deposition of type I collagen [Clare M. Lloyd, et al., J. Exp. Med. 1997, 185, 1371]. In addition, MCP-1−/− mice with induced nephrotoxic serum nephritis showed significantly less tubular damage than their MCP-1+/+ counterparts [Gregory H. Tesch, et al., J. Clin. Invest. 1999, 103, 73].
One study has demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating systemic lupus erythematosus. Crossing of MCP-1−/− mice with MRL-FAS.sup.1pr mice—the latter having a fatal autoimmune disease that is analogous to human systemic lupus erythematosus-results in mice with less disease and longer survival than the wildtype MRL-FAS.sup.1pr mice [Gregory H. Tesch, et al., J. Exp. Med. 1999, 190, 1813].
The MCP-1/CCR2 interaction in also relevant in the pathology of colitis as CCR-2−/− mice were protected from the effects of dextran sodium sulfate-induced colitis [Pietro G. Andres, et al., J. Immunol. 2000, 164, 6303].
One study has demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating alveolitis. When rats with IgA immune complex lung injury were treated intravenously with antibodies raised against rat MCP-1 (JE), the symptoms of alveolitis were partially alleviated [Michael L. Jones, et al., J. Immunol. 1992, 149, 2147].
The MCP-1/CCR2 interaction is also relevant in cancer. When immunodeficient mice bearing human breast carcinoma cells were treated with an anti-MCP-1 antibody, inhibition of lung micrometastases and increases in survival were observed [Rosalba Salcedo, et al., Blood 2000, 96, 34-40]. In particular MCP-1 is indicated in prostate cancer as described in details in PCT WO 2004/080273 to the present inventors.
Restinosis is yet another indication in which MCP-1 is involved. Mice deficient in CCR2 showed reductions in the intimal area and in the intima/media ratio (relative to wildtype littermates) after injury of the femoral artery [Merce Roque, et al. Arterioscler. Thromb. Vasc. Biol. 2002, 22, 554-559).
Other studies have provided evidence that MCP-1 is overexpressed in various disease states not mentioned above. These reports provide correlative evidence that MCP-1 antagonists could be useful therapeutics for such diseases. MCP-1 has been shown to be overexpressed in the intestinal epithelial cells and bowel mucosa of patients with inflammatory bowel disease [H. C. Reinecker, et al., Gastroenterology 1995, 108, 40; Michael C. Grimm, et al., J. Leukoc. Biol. 1996, 59, 804]. Two reports describe the overexpression of MCP-1 rats with induced brain trauma [J. S. King, et al., J. Neuroimmunol. 1994, 56, 127; Joan W. Berman, et al., J. Immunol. 1996, 156, 3017]. MCP-1 has also been shown to be overexpressed in rodent cardiac allografts, suggesting a role for MCP-1 in the pathogenesis of transplant arteriosclerosis [Mary E. Russell, et al. Proc. Natl. Acad. Sci. USA 1993, 90, 6086]. The overexpression of MCP-1 has been noted in the lung endothelial cells of patients with idiopathic pulmonary fibrosis [Harry N. Antoniades, et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5371]. Similarly, the overexpression of MCP-1 has been noted in the skin from patients with psoriasis [M. Deleuran, et al., J. Dermatol. Sci. 1996, 13, 228, and R. Gillitzer, et al., J. Invest. Dermatol. 1993, 101, 127]. Finally, a recent report has shown that MCP-1 is overexpressed in the brains and cerebrospinal fluid of patients with HIV-1-associated dementia [Alfredo Garzino-Demo, WO 99/46991].
Most chemokine antagonists reported to date are either neutralizing antibodies to specific chemokines or small molecule antagonists [Howard et al., Trend Biotechnol 14:46-51 (1996)].
Anti-MCP-1 antibodies have been used effectively in a number of mouse disease models as described above. However, a major problem associated with using antibodies to antagonize chemokine function is that they must be humanized before use in chronic human diseases. Furthermore, the ability of multiple chemokines to bind and activate a single receptor forces the development of a multiple antibody strategy or the use of cross-reactive antibodies in order to completely block or prevent pathological conditions.
Several small molecule antagonists of chemokine receptor function have been reported in the scientific and patent literature [White, J Biol Chem 273:10095-10098 (1998); Hesselgesser, J Biol Chem 273:15687-15692 (1998); Bright et al., BioorgMed Chem Lett 8:771-774 (1998); Lapierre, 26th Natl Med Chem Symposium, June 14-18, Richmond (Va.), USA (1998); Forbes et al., Bioorg Med Chem Lett 10:1803-18064 (2000); Kato et al., WO 97/24325; Shiota et al., WO 97/44329; Naya et al., WO 98/04554; Takeda Industries, JP 09-55572 (1998); Schwender et al., WO 98/02151; Hagmann et al., WO 98/27815; Connor et al., WO 98/06703; Wellington et al., U.S. Pat. No. 6,288,103]. The specificity of the chemokine receptor antagonists, however, suggests that inflammatory disorders characterized by multiple or redundant chemokine expression profiles will be relatively more refractory to treatment by these agents.
There is thus a widely recognized need for, and it would be highly advantageous to have, compositions and methods using same for treating CCR-2 associated diseases which are devoid of the above limitations.